A new fixative which primarily stabilizes carbohydrate moieties was developed for immunoelectron microscopy. It contains periodate, lysine and paraformaldehyde. Theoretically, the carbohydrates are oxidized by periodate and cross-linked by lysine. The fixative can preserve antigenicity as well as paraformaldehyde and ultrastructure as well as glutaraldehyde. Using this fixative and the peroxidase-labeled antibody technique, basement membrane antigen was localized within the cisternae of endoplasmic reticulum of parietal yolk sac cells and in extracellular basement membranes with adequate tissue successfully accomplished by conventional tissues fixed in glutaraldehyde and paraformaldehyde solutions.

A new method of conjugating horseradish peroxidase with proteins was developed. The c arbohydrate moiety of fluorodinitrobenzene-blocked peroxidase was oxidized with sodium periodate to form aldehyde groups. The peroxidase-aldehyde was then bound to free amino groups of proteins unidirectionally at high efficiencies. Peroxidase-labeled immuneglobulin retained its immunologic as well as enzymatic activities.

Peroxidase-labeled antibody method was used to localize the six hormones of the anterior pituitary gland of male rats both at the light and electron microscopic levels. Growth hormone (GH), adenocorticotropic hormone (ACTH), prolactin and thyrotropic hormone (TSH) were found in separate cells. Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were frequently found in the same cell. TSH cells were scarce and were located at the periphery of the gland. The anterior-ventral portion of the gland contained few or no GH cells, ACTH cells, prolactin cells and TSH cells, but was filled with gonadotropic cells. In an area near the intermediate lobe, GH cells, ACTH cells and TSH cells were not found. GH cells and prolactin cells may be identified in electron micrographs without the aid of immunocytochemistry; however, ACTH cells and TSH cells may not be distinguished by their ultrastructural characteristics alone. Gonadotropic cells may be identified but their hormone content cannot be determined. The positive identification of these latter four cell types requires immunocytochemical methods.

Enzymes, either acid phosphatase or horscradish pcroxidase, were conjugated to antibodics with bifunctional reagents. The conjugates, cnzymatically and immunologically active, werc employed in the immunohistochcmical localization of tissue antigens utilizing the reaction product of the enzymatic reaction as the marker. Tissues reacted with acid phosphataselabcled antibodies directed against basemcnt membrane werc stained for the enzyme with Gomori's method, and those reacted with peroxidase-labcled antibody were stained with Karnovsky's method. The reaction products of the enzymes localized in the basement membrane. Unlike thc preparations of the fluorescent antibody technique, enzyme-labelcd antibody prcparations were permanent, could be observed with an ordinary microscope, and could be examined with the electron microscope. In the latter, specific localization of antibody occurred in the basement membrane and in the endoplasmic reticulum of cells known to synthesize basemcnt mcmbrane antigens. The method is sensitive bccause of the amplifying effect of the enzymatic activity. The ultrastructural preservation and localization were better with acid phosphatase-labeled antibody than with peroxidase-labclcd antibody, but acid phosphatase conjugated antibody was unstable and difficult to prepare. Peroxidaseantibody conjugatcs were stable and could bc stored for several months at 4°C, or indcfiniely in a frozen state.

The peroxidase-labeled antibody method was modified to localize multiple tissue antigens in a single histologic section.